Structural studies of HCN channels in health and disease

项目摘要 本项目的目标是了解HCN通道的门控是如何通过位于 蛋白质的细胞质和细胞内部分。HCN通道调节心脏的起搏， 大脑中神经元的兴奋性。它们具有电压门控性K+通道的一般结构，但 在膜超极化时激活并传导向内的去极化电流。重要的是他们 门控通过cAMP与细胞内环核苷酸结合结构域（CNBD）的直接结合来调节 位于蛋白质的C末端部分。最近的结构数据还显示， 第二个高度结构化的结构域，位于蛋白质的细胞内N-末端部分，紧接着 第一跨膜结构域（HCN结构域）。在这项研究中，我们将使用结构和 确定HCN结构域在通道门控调节中的潜在作用的功能方法， 以及C-末端CNBD和N-末端HCN结构域之间相互作用的性质。为不同 HCN亚型（HCN 1 -4）表现出显着不同的生物物理特性，我们将利用这些差异来 确定可能有助于调节HCN通道门控的关键接触点和残基。目标1： 将使用冷冻电子显微镜（cryoEM）来确定HCN 4亚型的结构， 和不存在cAMP，并将其特征与HCN 1的可用cryoEM结构进行比较。在目标2中，我们将 在存在干扰cAMP介导的促进作用的配体的情况下解析HCN 4的结构， 通道开放（辅助亚基TRIP 8b，联苯脲化合物BPU），并使用结构数据 通过目标1和2获得的信息，为旨在测试和解释任何推断的功能实验提供信息。 结构/功能关系模型。最后，在目标3中，我们将使用cryoEM和 功能研究，研究遗传性癫痫患者中发现的HCN 1和HCN 4突变， 心律失常

Project Summary The goal of this project is to understand how the gating of HCN channels is regulated by domains located in the cytoplasmic, intracellular portion of the protein. HCN channels regulate pacemaking in the heart, and neuronal excitability in the brain. They have the general structure of voltage-gated K+ channels, but are activated upon membrane hyperpolarization and conduct an inward, depolarizing current. Importantly, their gating is modulated by the direct binding of cAMP to an intracellular cyclic-nucleotide binding domain (CNBD) located in the C-terminal portion of the protein. Recent structural data have also revealed the presence of a second, highly structured domain in the intracellular N-terminal portion of the protein, immediately preceding the first transmembrane domain (HCN domain). In this study, we will use a combination of structural and functional approaches to determine the potential role of the HCN domain in the modulation of channel gating, and the nature of the interactions between the C-terminal CNBD and N-terminal HCN domain. As different HCN isoforms (HCN1-4) exhibit markedly distinct biophysical properties, we will exploit these differences to identify key contacts and residues that may contribute to modulate the gating of HCN channels. In Aim 1, we will use cryo-electron microscopy (cryoEM) to determine the structure of the HCN4 isoform, in the presence and absence of cAMP, and compare its features to the available cryoEM structure of HCN1. In Aim 2, we will resolve the structure of HCN4 in the presence of ligands that interfere with the cAMP-mediated facilitation of channel opening (auxiliary subunit TRIP8b, biphenylurea compound BPU), and use the structural data acquired through Aims 1 and 2 to inform functional experiments designed to test and interpret any inferred model of structure/function relation. Finally, in Aim 3, we will use a similar combination of cryoEM and functional studies to study select HCN1 and HCN4 mutations found in patients with genetic epilepsy and cardiac arrhythmias.